RNA interference (RNAi) refers to the process of sequence-specific post transcriptional gene silencing mediated by small interfering RNAs (siRNA) (Fire et al., 1998, Nature, 391, 806-11). Long double stranded RNA (dsRNA) in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the long dsRNA into short pieces of siRNA (Bernstein et al., 2001, Nature, 409, 363-6). siRNAs derived from dicer activity are typically about 21-23 nucleotides in length and include duplexes of about 19 base pairs.
The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Nature, 411, 494-498).
siRNA mediated RNAi has been studied in a variety of systems. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877-88). RNAi technology has been used in mammalian cell culture, where a siRNA-mediated reduction in gene expression has been accomplished by transfecting cells with synthetic RNA oligonucleotides (Caplen et al., 2001, Proc. Natl. Acad. Sci., U.S.A., 98, 9742-7; Elbashir et al., 2001, Nature, 411, 494-8). The ability to use siRNA-mediated gene silencing in mammalian cells combined with the high degree of sequence specificity allows RNAi technology to be used to selectively silence expression of mutant alleles or toxic gene products in dominantly inherited diseases, including neurodegenerative diseases. Several neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, Spinocerebellar Ataxia Type 1, Type 2, and Type 3, and dentatorubral pallidoluysian atrophy (DRLPA), have proteins identified that are involved in the overall pathogenic progression of the disease.
siRNA-mediated gene silencing of mutant forms of human ataxin-3, Tau and TorsinA, genes which cause neurodegenerative diseases such as spinocerebellar ataxia type 3, frontotemporal dementia and DYTI dystonia respectively, has been demonstrated in cultured cells (Miller et al. 2003, Proc. Natl. Acad. Sci., U.S.A., 100, 7195-7200; Gouzales-Alegre et al., 2003, Ann. Neurol. 53, 781-7).
α-synuclein (α-syn) is involved in the pathogenesis of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), the Lewy body variant of Alzheimer's disease (LBVAD), multiple systems atrophy (MSA), and neurodegeneration with brain iron accumulation type-1 (NBIA-1), as well as sleep and other disorders. Common to all of these diseases, termed synucleinopathies, are proteinaceous insoluble inclusions in the neurons and the glia which are composed primarily of α-syn.
α-syn is part of a large family of proteins including β- and γ-synuclein and synoretin. α-syn is expressed in the normal state associated with synapses and plays a role in neural plasticity, learning and memory. Mutations in the human α-syn (h-α-syn) gene that enhance the aggregation of α-syn have been identified (alanine to threonine substitution at position 53 (A53T) and alanine to proline at position 30 (A30) and are associated with rare forms of autosomal dominant forms of PD. Altered h-α-syn function triggers neurodegenerative processes associated with PD such as the selective loss of dopaminergic neurons in the substantia nigra pars compacta leading to substantial depletion of dopamine in the striatum resulting in severe motor impairment (Dawson et al., 2002, Nat. Neurosci., November; 5 Suppl: 1058-61). Abnormal accumulation of wild-type or mutant α-syn impairs proteasome function, interferes with vesicular dopamine storage, renders endogenous dopamine toxic, and contributes to mitochondrial dysfunction (Polymeropoulos, M., 2000, Ann. NY. Acad. Sci., 920, 28-32, Lotharius et al., 2002, Nature Reviews Neurosci. 3, 932-42).
A need exists for a siRNA-mediated gene silencing methods and systems for silencing α-syn and its family members.